Glycolipids, a group of amphipathic compounds that structurally consist of a sugar chain (monosaccharide or oligosaccharide) bound to an aglycone, are important cellular membrane components known to participate in various cellular events mediating physiological processes such as the cell-cell recognition, antigenicity, and cell growth regulation (Hakomori, Annu. Rev. Biochem., 50: 733-764, 1981; Makita and Taniguchi, Glycolipid (Wiegandt, ed.) pp 59-82, Elsevier Scientific Publishing Co., New York, 1985). Because there are no known enzymes that can universally transfer a saccharyl residue to a an aglycone (e.g., ceramide or sphingosine), synthesis of glycolipids usually requires a multi-step complex process that has the disadvantages of high cost and low yield.
Endoglycoceramidase (EC3.2.1.123), an enzyme first isolated from the Actinomycetes of Rhodococcus strain (Horibata, J. Biol. Chem. May 2004 10.1094/jbc.M401460200; Ito and Yamagata, J. Biol. Chem., 261: 14278-14282, 1986), hydrolyzes the glycoside linkage between the sugar chain and the ceramide in glycolipids to produce intact monosaccharide or oligosaccharide and ceramide. To this date, several more endoglycoceramidases have been isolated and characterized (see e.g., Li et al., Biochem. Biophy. Res. Comm., 149: 167-172, 1987; Ito and Yamagata, J. Biol. Chem., 264: 9510-9519, 1989; Zhou et al., J. Biol. Chem., 264: 12272-12277, 1989; Ashida et al., Eur. J. Biochem., 205: 729-735, 1992; Izu et al., J. Biol. Chem., 272: 19846-19850, 1997; Horibata et al., J. Biol. Chem., 275:31297-31304, 2000; Sakaguchi et al., J. Biochem., 128: 145-152, 2000; and U.S. Pat. No. 5,795,765). The active site of endoglycoceramidases has also been described by Sakaguchi et al., Biochem. Biophy. Res. Comm., 260: 89-93, 1999, as including a three amino acid segment of Asn-Glu-Pro, among which the Glu residue appears to be the most important to the enzymatic activity.
Endoglycoceramidases are also known to possess an additional transglycosylation activity, which is much weaker than the hydrolytic activity (Li et al., J. Biol. Chem., 266:10723-10726, 1991; Ashida et al., Arch. Biochem. Biophy., 305:559-562, 1993; Horibata et al., J. Biochem., 130:263-268, 2001). This transglycosylation activity has not yet been exploited to synthesize glycolipids, because the far more potent hydrolytic activity of the enzyme counteracts this synthetic activity by quickly hydrolyzing newly made glycolipid.
In view of the deficiencies of the current methods for chemically synthesizing glycosphigolipids, a method that relies on the substrate specificity of a synthetic endoglycoceramidase would represent a significant advance in the field of saccharide (glycolipid) synthesis. The present invention provides such a synthetic endoglycoceramidase (“endoglycoceramide synthase”) and methods for using this new enzyme.